• Archives of Pharmacal Research
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• v.24 no.2
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• pp.126-135
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• 2001

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of $DiOC_6$/ demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (${\blacktriangle}{\Psi}_m$). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector caspase-3, Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, NDCA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDCA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent, and caspase-8 activation may be upstream of the mitochondrial events. In summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.

• BMB Reports
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• v.46 no.2
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• pp.119-123
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• 2013

Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), an endogenous neurotoxin, is known to perform a role in the pathogenesis of Parkinson's disease (PD). In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with salsolinol. When cytochrome c was incubated with salsolinol, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in salsolinol-treated cytochrome c. Salsolinol also led to the release of iron from cytochrome c. Reactive oxygen species (ROS) scavengers and iron specific chelator inhibited the salsolinol-mediated cytochrome c modification and carbonyl compound formation. It is suggested that oxidative damage of cytochrome c by salsolinol might induce the increase of iron content in cells, subsequently leading to the deleterious condition which was observed. This mechanism may, in part, provide an explanation for the deterioration of organs under neurodegenerative disorders such as PD.

• BMB Reports
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• v.37 no.4
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• pp.445-453
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• 2004

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.

• Biomedical Science Letters
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• v.12 no.2
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• pp.57-63
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• 2006

Deazaadenosine analogs such as 3-deazaadenosine (DZA), 3-deazaaristeromycin (DZAri) and ara-3-deazaadenine (DZAra-A) were developed as inhibitors of S-adenosylhomocysteine (Ado-Hcy) hydrolase (EC 3.3.1.1). These analogs were reported to induce apoptosis in human and murine leukemic cells. But, the mechanism involved in this apoptosis was not clarified yet. In the present study, we analyze the apoptosis induced by deazaadenosine analogs in human cervival cancer cell line, HeLa and the effect of Bcl-2 on this apoptosis. Whereas neither DZAri nor DZAra-A showed inhibitory effect on HeLa cell growth, DZA induced apoptosis in HeLa cells accompanied by cytochrome c release and activation of various caspases such as caspase-2,-8,-9 and -3. In HeLa-bcl-2 cell line, a stable transfectant of HeLa cell to overexpress Bcl-2, cytochrome c release, activation of all these caspases and the resulted apoptosis by DZA were completely prevented. By in vitro assay of cytochrome c release, in addition, DZA induced cytochrome c release from purified mitochondria of HeLa-pcDNA3 cells, but not HeLa-bcl-2 cells, even in the absence of cytosolic fraction. Therefore, it can be suggested that DZA might damage directly mitochondria leading to activate intrinsic pathway of caspase and thus induce apoptosis. DZA-induced apoptosis in HeLa cells may be in a bcl-2-inhibitable manner and irrelative of Ado-Hcy hydrolase.

• BMB Reports
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• v.38 no.5
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• pp.619-623
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• 2005

The efficacy of chemotherapeutic agents on tumor cells has been shown to be modulated by tumor suppressor gene p53 and its target genes such as Bcl-2 family members (Bax, Noxa, and PUMA). However, various chemotherapeutic agents can induce cell death in tumor cells that do not express the functional p53, suggesting that some chemotherapeutic agents may induce cell death in a p53-independent pathway. Here we showed that etoposide can induce the similar degree of cell death in p53-deficient HCT 116 cells, whereas 5'-FU-mediated cell death is strongly dependent on the existence of functional p53 in HCT 116 cells. Further, we provide the evidence that etoposide can induce the cytochrome c release from isolated mitochondria, and etoposide-induced cytochrome c release is not accompanied with the large amplitude swelling of mitochondria. These data suggest that etoposide can directly induce the mitochondrial dysfunction irrespective of p53 status, and it may, at least in part, account for the p53-independent pathway in cell death induced by chemotherapeutic agents.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2343-2347
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• 2013

Objective: This investigation aimed to determine effects of celecoxib on the cell cycle kinetics of the gastric cancer cell line MGC803 and the mechanisms involved by assessing expression of cytochrome C and caspase-9 at the protein level. Methods: Cell proliferation of MGC803 was determined by MTT assay after treatment with celecoxib. Apoptosis was assessed using fluorescence staining and cell cycle kinetics by flow cytometry. Western blotting was used to detect the expression of caspase-9 protein and of cytochrome C protein in cell cytosol and mitochondria. Results: Celecoxib was able to restrain proliferation and induce apoptosis in a dose- and time-dependent manner, inducing G0/G1 cell cycle arrest, release of cytochrome C into the cytosol, and cleavage of pro-caspase-9 into its active form. Conclusion: Celecoxib can induce apoptosis in MGC803 cells through a mechanism involving cell cycle arrest, mitochondrial cytochrome C release and caspase activation.

• Archives of Pharmacal Research
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• v.28 no.1
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• pp.87-92
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• 2005

The cyclin-dependent kinase inhibitor$p27^{kip1}$(p27) has been implicated in the regulation of cell cycle and apoptosis. Recently, we have demonstrated that ceramide induces apoptotic cell death associated with increase in the level of p27 in HL-60 cells. In the present study, we showed that overexpression of p27 increases ceramide-induced apoptotic cell death in HL-60 cells. Furthermore, overexpression of p27 accelerated DNA fragmentation, PARP cleavage and cytochrome c release induced by ceramide. In addition, ceramide induced Sax expression independent of p27. These findings indicate that enhanced effect on apoptosis by p27 is associated with mitochondrial signaling which involves cytochrome c release.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.167.1-167.1
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• 2003

Here we show that panaxadiol, a ginseng saponin with a dammarane skeleton, induces acute apoptotic cell death in human hepatoma SK-HEP-1 cells as evidenced by analysis of DNA fragmentation, caspase activation, and changes in cell morphology. The kinetic study showed that panaxadiol-induced apoptosis is associated with depolarization of mitochondrial membrane potential and cytochrome c release. Sequential activations of caspases-depolarization of mitochondrial membrane potential and cytochrome c release. Sequential activations of caspases-9, and -3, or -7, but not of caspase 8 coincide well in a time dependent manner with mitochondrial membrane depolarization and cytochrome c release from mitochondria during apoptosis of SK-HEP-1 cells induced by treatment with panaxadiol. (omitted)

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.319.1-319.1
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• 2002

Yomogin. an eudesmane sesquiterpene isolated from Artemisia princeps, was found to induce apoptosis in human promyelocytic leukaemia, HL -60 cell with characteristic apoptotic features like nuclear condensation, apoptotic body formation, flipping of membrane phosphatidylserine, release of mitochondrial cytochrome c and caspase-8. -9. and -3 activation. Furthermore. early yomogin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAd fmk and preceded loss of mitochondrial membrane potential. The results suggest that induction of apoptosis by yomogin may provide a pivotal mechanism for their cancer chemopreventive function.

• Animal cells and systems
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• v.7 no.1
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• pp.1-9
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• 2003

Programmed cell death, or apoptosis, is one of the most studied areas of modern biology. Apoptosis is a genetically regulated process, which plays an essential role in the development and homeostasis of higher organisms. Mitochondria, known to play a central role in regulating cellular metabolism, was found to be critical for regulating apoptosis induced under both physiological and pathological conditions. Mitochondria are a major source of reactive oxygen species (ROS) but they can also serve as its target during the apoptosis process. Release of apoptogenic factors from mitochondria, the best known of which is cytochrome c, leads to assembly of a large apoptosis-inducing complex called the apoptosome. Cysteine pretenses (called caspases) are recruited to this complex and, following their activation by proteolytic cleavage, activate other caspases, which in turn target for specific cleavage a large number of cellular proteins. The redox regulation of apoptosis during and after cytochrome c release is an area of intense investigation. This review summarizes what is known about the biological role of ROS and its targets in apoptosis with an emphasis on its intricate connections to mitochondria and the basic components of cell death.